Trp-notrp-notrp-notrp-notrp-notrp-notrp-no: Rapid, multiplexed, sensitive and specific identification and quantitative detection of nitric oxide (NO) are in great demand in biomedical science. Herein, a novel multifunctional probe based on the intramolecular LRET (luminescence resonance energy transfer) strategy, , was designed for the highly sensitive and selective ratiometric and luminescence lifetime detection of lysosomal NO. Before reaction with NO, the emission of the rhodamine moiety in is switched off, which prevents the LRET process, so that the probe emits only the long-lived Tb3+ luminescence. However, upon reaction with NO, accompanied by the turn-on of rhodamine emission, the LRET from the Tb3+-complex moiety to rhodamine moiety occurs, which results in a remarkable increase of the rhodamine emission and decrease of the Tb3+ emission. After the reaction, the intensity ratio of the rhodamine emission to the Tb3+ emission, I565/I540, was found to be 28.8-fold increased, and the dose-dependent enhancement of the I565/I540 value showed a good linearity upon the increase of NO concentration. In addition, a dose-dependent luminescence lifetime decrease was distinctly observed between the average luminescence lifetime of the probe and NO concentration, which provides a &sim;10-fold contrast window for the detection of NO. These unique properties allowed to be conveniently used as a time-gated luminescence probe for the quantitative detection of NO using both luminescence intensity ratio and luminescence lifetime as signals. The applicability of for ratiometric time-gated luminescence imaging of NO in living cells was investigated. Meanwhile, dye co-localization studies confirmed a quite precise distribution of in lysosomes by confocal microscopy imaging. Furthermore, the practical applicability of was demonstrated by the visualization of NO in Daphnia magna. All of the results demonstrated that could serve as a useful tool for exploiting and elucidating the function of NO at sub-cellular levels with high specificity, accuracy and contrast.

sch1: Structure of the probe TRP-NO and its luminescence response reaction with NO.

Mentions:
In this work, a unique multifunctional probe, TRP-NO, for the sensitive and specific recognition of lysosomal NO with both ratiometric and luminescence lifetime detection modes has been successfully designed and synthesized based on the intramolecular LRET (luminescence resonance energy transfer) mechanism from a luminescent Tb3+ complex (LTC) to rhodamine (5-carboxytetramethylrhodamine, CTMR). Before reaction with NO, since the spirolactam derivative of rhodamine moiety in the probe is non-fluorescent, TRP-NO emits only the strong Tb3+ luminescence. While upon reaction with NO, the spirolactam-ring of the rhodamine moiety is opened, which results in the recovery of the LRET process, meanwhile the time-gated luminescence intensities of LTC and CTMR moieties are remarkably decreased and increased, respectively. The intensity ratio of CTMR/LTC emissions showed a good linearity against the NO concentration. Interestingly, the average luminescence lifetime of TRP-NO also showed dose-dependent changes with the variation of the NO concentration, which enabled “luminescence lifetime” to be used as a signal responding to the change of NO concentration. On the basis of these findings, a multifunctional probe equipped with both ratiometric time-gated luminescence intensity and luminescence lifetime responses to NO, TRP-NO, was developed. In combination with a true-color time-gated luminescence microscope, TRP-NO was successfully used for the imaging of NO in HepG2 cells and Daphnia magna. In addition, dye co-localization studies testified to a quite precise distribution of TRP-NO in lysosomes by the confocal microscopy imaging. Scheme 1 shows the structure of TRP-NO and its luminescence response reaction with NO.

sch1: Structure of the probe TRP-NO and its luminescence response reaction with NO.

Mentions:
In this work, a unique multifunctional probe, TRP-NO, for the sensitive and specific recognition of lysosomal NO with both ratiometric and luminescence lifetime detection modes has been successfully designed and synthesized based on the intramolecular LRET (luminescence resonance energy transfer) mechanism from a luminescent Tb3+ complex (LTC) to rhodamine (5-carboxytetramethylrhodamine, CTMR). Before reaction with NO, since the spirolactam derivative of rhodamine moiety in the probe is non-fluorescent, TRP-NO emits only the strong Tb3+ luminescence. While upon reaction with NO, the spirolactam-ring of the rhodamine moiety is opened, which results in the recovery of the LRET process, meanwhile the time-gated luminescence intensities of LTC and CTMR moieties are remarkably decreased and increased, respectively. The intensity ratio of CTMR/LTC emissions showed a good linearity against the NO concentration. Interestingly, the average luminescence lifetime of TRP-NO also showed dose-dependent changes with the variation of the NO concentration, which enabled “luminescence lifetime” to be used as a signal responding to the change of NO concentration. On the basis of these findings, a multifunctional probe equipped with both ratiometric time-gated luminescence intensity and luminescence lifetime responses to NO, TRP-NO, was developed. In combination with a true-color time-gated luminescence microscope, TRP-NO was successfully used for the imaging of NO in HepG2 cells and Daphnia magna. In addition, dye co-localization studies testified to a quite precise distribution of TRP-NO in lysosomes by the confocal microscopy imaging. Scheme 1 shows the structure of TRP-NO and its luminescence response reaction with NO.

Trp-notrp-notrp-notrp-notrp-notrp-notrp-no: Rapid, multiplexed, sensitive and specific identification and quantitative detection of nitric oxide (NO) are in great demand in biomedical science. Herein, a novel multifunctional probe based on the intramolecular LRET (luminescence resonance energy transfer) strategy, , was designed for the highly sensitive and selective ratiometric and luminescence lifetime detection of lysosomal NO. Before reaction with NO, the emission of the rhodamine moiety in is switched off, which prevents the LRET process, so that the probe emits only the long-lived Tb3+ luminescence. However, upon reaction with NO, accompanied by the turn-on of rhodamine emission, the LRET from the Tb3+-complex moiety to rhodamine moiety occurs, which results in a remarkable increase of the rhodamine emission and decrease of the Tb3+ emission. After the reaction, the intensity ratio of the rhodamine emission to the Tb3+ emission, I565/I540, was found to be 28.8-fold increased, and the dose-dependent enhancement of the I565/I540 value showed a good linearity upon the increase of NO concentration. In addition, a dose-dependent luminescence lifetime decrease was distinctly observed between the average luminescence lifetime of the probe and NO concentration, which provides a &sim;10-fold contrast window for the detection of NO. These unique properties allowed to be conveniently used as a time-gated luminescence probe for the quantitative detection of NO using both luminescence intensity ratio and luminescence lifetime as signals. The applicability of for ratiometric time-gated luminescence imaging of NO in living cells was investigated. Meanwhile, dye co-localization studies confirmed a quite precise distribution of in lysosomes by confocal microscopy imaging. Furthermore, the practical applicability of was demonstrated by the visualization of NO in Daphnia magna. All of the results demonstrated that could serve as a useful tool for exploiting and elucidating the function of NO at sub-cellular levels with high specificity, accuracy and contrast.